The Nobel Prize in Physiology or The Nobel Prize in Physiology or Medicine 2004Medicine 2004

Discoveries of odorant Discoveries of odorant receptors and the receptors and the

organization of the olfactory organization of the olfactory system system

醫學一 B9602084 潘 岳 B9602091 謝仁峰

B9602086 鄭尉佐 B9602098 羅緯麟

B9602088 盧柏嘉

Discoveries of odorant receptors and the Discoveries of odorant receptors and the organization of the olfactory system organization of the olfactory system

Richard AxelRichard Axel Linda B.BuckLinda B.Buck

Columbia University New York, NY, USA

Fred Hutchinson Cancer Research Center Seattle, WA, USA

Three assumptionsThree assumptions

Receptors are G-protein-coupled receptors. Receptors are G-protein-coupled receptors. (GPCRs) (GPCRs)

Diverse receptors encoded by multigene famiDiverse receptors encoded by multigene familyly

Receptor expression restricted to olfactory eReceptor expression restricted to olfactory epitheliumpithelium

Homogenize the epithelium cellsHomogenize the epithelium cells

‧RNA

Many double-stranded cDNAsAdd RNA and reverse transcriptase

Reverse transcriptase Reverse transcriptase cDNAcDNA

Specific primerSpecific primer GPCRs familyGPCRs family PCRPCR primer

II VII

Add primers anneal to conserved regions(II VII) of the GPCR family

Different species cDNA exist

M

3600

600

1700

900

1200

1500

300

ElectrophoresisElectrophoresis ：： initiationinitiation

M

3600

600

1700

900

1200

1500

300

ElectrophoresisElectrophoresis ：： resultresult

M

3600

600

1700

900

1200

1500

300

Simple cDNAs Mixed-up cDNAs

Add restriction enzyme

Northern blotNorthern blot

MaterialRNA from epithlium/brain/spleenlabeled PCR13 products as probeResult Brain & Spleen :no hybridization

Expression is restricted to olfactory epithelium Demonstrate assumption 3Obtain full-length c-DNA

Protein sequenceProtein sequence

N-terminus

C-terminus

various

Related to ligand recognition

Experiment summaryExperiment summary

DNA sequencingDNA sequencing

Assumption 1 :GPCRAssumption 1 :GPCR

Assumption 2 :Multigene→various receptorAssumption 2 :Multigene→various receptor

Northern blottingNorthern blotting

Assumption 3 :Expression locationAssumption 3 :Expression location

protein sequenceprotein sequence

The family of olfactory The family of olfactory genes in humangenes in human

1.1. Over 1000 genesOver 1000 genes

2.2. About 400 genes functionAbout 400 genes function

; 600 genes not function; 600 genes not function

Olfactory receptor genesOlfactory receptor genes

Olfactory sensory

neuron

Species differencesSpecies differences

Sensitivity

High Low

Olfactory Sensory Neuron(OSN)Olfactory Sensory Neuron(OSN)

Olfactory

Sensory neuron

A large family of odorant receptors A large family of odorant receptors One neuron cell expresses

only one type of receptor

7 transmenbrane domains

Nitrite group in extracellular space

Conformational change

Olfactory

Sensory

neuron

Combinatorial receptor codesCombinatorial receptor codes

Sensory transductionSensory transduction

SensorySensory transductiontransduction

Olfactory BulbOlfactory Bulb

Olfactory Receptor Olfactory Receptor V.S.V.S. Olfactory glomerulus Olfactory glomerulus

Map of Olfactory BulbMap of Olfactory Bulb

前 後

QQ ：： Human has about 400 types of receptHuman has about 400 types of receptors, but how do they discriminate about teors, but how do they discriminate about ten thousands odorants?n thousands odorants?

Combinatorial receptor codes experimentCombinatorial receptor codes experiment

100

Fura-2

10

1

Combinatorial receptor codesCombinatorial receptor codes

Multigene family

Receptor

express

Functional genes： pseudogenes

400： 600

Combination

Different ordor

recognized

Thank YouThank You ！！